Amyloid Filaments in Inclusion Body Myositis Novel Findings Provide Insight Into Nature of Filaments

Abstract

body myositis (IBM) represents a serious debilitating disease of muscle without identifiable cause or treatment.

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Muscle biopsy specimens have characteristic rimmed vacuoles, varying degrees of inflammation, and, most importantly, cytoplasmic and intranuclear filamentous inclusions of unknown composition. Fresh-frozen sections of muscle biopsy specimens from 24 IBM cases were stained with Congo red dye (pH, 10.5 to 11.0). Control biopsy specimens included polymyositis, dermatomyositis, hereditary vacuolar myopathies of unknown cause, acid maltase deficiency, distal myopathy, oculopharyngeal dystrophy, and chloroquine myopathy. Sections were also immunostained with antibody to transthyretin, human P component, and immunoglobulin light chains. In the vacuolated fibers in IBM, amyloidogenic green-birefringent deposits were seen. Some deposits were delicate and wispy appearing, and others were pl aque\x=req-\ like. The size of deposits varied, measuring 1 \m=x\2 to 8 \g=m\m, and rarely up to 20 \g=m\m in length. The number of amyloid-positive fibers correlated with the number of vacuolated fibers. Similar deposits were seen in one case of distal myopathy and one hereditary vacuolar myopathy. Other control cases were negative for amyloid deposits. Antibody staining for known amyloidogenic proteins was negative. This study demonstrates that the filaments in IBM share properties with amyloid proteins. The location implies that this amyloid material is formed intracellularly, rather than having a systemic derivation. The association of amyloid deposits with autophagic vacuoles in IBM raises the likely possibility that the filaments represent a modification of a normal protein within an acidic degradative vacuolar compartment. An alternative possibility, considering the shared properties of IBM filaments and prions (which include size and amyloidogenic properties), is that IBM represents a human prion disease. Further studies will be required to resolve the nature of the amyloidogenic filaments in IBM, but these findings may provide clues to the pathogenesis of this poorly understood disorder. In addition, the findings should facilitate the clinical diagnosis of IBM.

"1 He Term Inclusion Body Myositis

(IBM) was coined in 1971 by Yunis and Samaha. ' They described a disorder resembling polymyositis but distinctive because of the vacuolated muscle fibers and nuclear and cytoplasmic fibrillary inclusions. Controversy concerning the specificity of the histopathologic fea-(Arch Neurol. 1991 ;48:1229-1234) tures of inclusion body myositis delayed its general acceptance as a distinct dis¬ order. Subsequent reports24 have un¬ equivocally established this as a welldefined entity. The disorder slightly predominates in male individuals and usually begins after age 50 years, al¬ though no age groups have been entire¬ ly excluded.0 The diagnosis of IBM rests clearly on changes revealed by muscle biopsy. The characteristic features in¬ clude the presence of single or multiple, subsarcolemmal or centrally placed rimmed vacuoles. The vacuoles are lined with granular material that, by use of electron microscopy, presents features of autophagia composed of whorls of membranous material and myelin figures, as well as amorphous debris.2,4 In addition, endomysial in¬ flammation occurs to varying degrees, characterized by focal invasion of nonnecrotic muscle fibers by a 2:1 ratio of predominantly CD8+ cytotoxic/ suppressor cells to macrophages.8 The definitive diagnosis of IBM rests on electron microscopic findings of fila¬ mentous inclusions.1,2,4 The filaments are not membrane bound, but they often occur adjacent to the vacuoles. The diameter of the filaments has been re¬ ported to vary from 10 to 21 nm."

The cause of IBM remains enigmatic. The significance of the filamentous in¬ clusions represents a subject of debate. In 1967, Chou7 described a myxoviruslike structure in a case of chronic polymyositis. In retrospect, this repre¬ sented the first description of the fila¬ ments occurring in IBM. Subsequent work by Chou8 emphasized that these inclusions reacted with antibodies raised against mumps virus antigens. This finding was not confirmed by other investigators9,10 unable to demonstrate specific binding of anti-mumps antibod¬ ies in IBM.

The study presented herein provides new insight into the nature of IBM fila¬ ments. The findings strongly indicate that the filaments share histochemical properties with amyloid proteins, but the intracellular location distinguishes them from the more common types of systemic amyloid.11,12 The close associa¬ tion of amyloidogenic filaments to autophagic vacuoles in IBM raises the possi¬ bility of accumulation of an altered protein. Alternatively, properties com-mon to both IBM filaments and prion proteins suggest a possible relationship to the human prion disorders. 13~16

Muscle Specimens

All muscle specimens were embedded in gum tragacanth and frozen on wooden chucks in isopentane cooled in liquid nitrogen imme¬ diately on removal. In each case, 8to 10-pm sections were obtained for a battery of histo¬ chemical stains to establish the initial diagno¬ sis. Thereafter, the blocks were stored at -70°C until further use. A diagnosis of IBM was established in 24 samples obtained from three different muscle groups (biceps, = 16; deltoid, = 6; and quadriceps, = 2). All of these biopsy specimens demonstrated com¬ mon light microscopic features, including rimmed vacuoles, varying degrees of endo¬ mysial inflammation, and scattered angular fibers.4 To be included in the study, an abso¬ lute requirement was the presence of typical cytoplasmic or intranuclear filaments fulfill¬ ing previously described criteria. Addi¬ tional muscle samples studied included poly¬ myositis (n = 13) and dermatomyositis (n = 19). These patients all fulfilled the crite¬ ria of Bohan and Peter.1' A variety of other myopathies were also examined, including biopsy specimens taken from patients with oculopharyngeal muscular dystrophy (n = 4), acid maltase deficiency (n = 2), chloroquine myopathy (n = 1), distal myopathy (n = 1), periodic paralysis (n = 1), hereditary vacuo¬ lar myopathies of unknown cause (n = 5), Du¬ chenne muscular dystrophy (n = 2), and phosphogly cerate mutase deficiency (n = 1).

Congo Red Staining

A modification of the previously described alkaline Congo red method18 provided the most specific staining. Adjustment ofthe dif¬ ferentiating and Congo red solutions to a pH of 10.5 to 11.0 reduced background staining, especially in comparison with the staining methods for amyloid by Bennhold,1' Highman,2" and Lieb. ] Fresh-frozen 10to 12-µ muscle biopsy sections were placed on methanol-cleaned slides. All solutions used for staining were prepared at the time of use and then dis¬ carded. Sections were placed in Mayer's he¬ matoxylin22 for 10 minutes, rinsed in three changes of distilled water, transferred to a solution containing 80% ethanol saturated with sodium chloride, and adjusted to a pH of 10.5 to 11.0 with 1% sodium hydroxide. The sections were then stained for 1 hour with Congo red dye (0.2 g in 100 mL of 80% ethanol saturated with sodium chloride and adjusted to a pH of 10.5 to 11.0 with 1% sodium hy¬ droxide). Following staining, sections were dehydrated in 95% ethanol and absolute eth¬ anol, cleared in xylene, and mounted in a synthetic mounting medium (Permount).

Potassium permanganate method, as de¬ scribed by Wright et al, ' was used for preincubation in some cases.

Immune Staining

For antibody localization, 8-to 10-µ sec¬ tions of fresh-frozen muscle biopsy speci¬ mens were fixed in acetone for 1 minute. Antibody against human transthyretin or human component (ATAB, Scarborough, Mass) was incubated for 1 hour at room tem¬ perature at a 1:50 dilution (matched for pro¬ tein content with control serum samples). Sections were washed in phosphate-buffered saline and incubated for 1 hour at room tem¬ perature with biotinylated anti-sheep IgG (Jackson Immunoresearch) at a 1:200 dilu¬ tion. Fluorescein-conjugated avidin (EY Labs, San Mateo, Calif) at a 1:200 dilution was used to visualize antibody localization.

Electron Microscopy

For ultrastructure, muscle specimens were clamped in situ, removed, and placed in a solution containing 3% glutaraldehyde in 0.1 mol/L phosphate buffer for 30 minutes. The samples were dissected from the clamp, and 1to 2-mm cubes were kept in glutaralde¬ hyde for 5 hours, postfixed for 1 hour in 2% osmium tetroxide and 0.1 mol/L phosphate buffer, dehydrated in graded ethanol, and embedded in low-viscosity embedding me¬ dia. Sections, 1 µ thick, stained with toluidine blue were examined under light micros¬ copy, and appropriate blocks were selected for electron microscopy examination.

Muscle Fiber Quantitation

The frequency of amyloid-positive fibers was determined in 15 IBM cases selected on the basis of random number assignment. Amyloid-positive fibers and total number of vacuolated fibers were counted in Congo redstained sections under polarized light. Adja¬ cent fresh-frozen sections of muscle stained with adenosine triphosphate at a pH of 9.4 4 were projected onto a video screen with an overlaid grid to provide orientation. All mus¬ cle fibers in the section were counted.

Patient Population

Twenty-four consecutive cases of IBM fulfilled the previously described light and electron microscopic crite¬ ria.1,2,4 The clinical features did not differ in any way from the largest population of patients recently described.4 There were 12 men and 12 women. The mean age at diagnosis was 64.3 years (range, 32 to 76 years), with only three patients aged below 60 years (two men aged 30 and 38 years; one woman aged 57 years). Onset of symptoms prior to diagnosis varied from 1.5 to 15 years. Only one patient had an associated connective tis¬ sue disease, rheumatoid arthritis. Oth¬ er disorders included diabetes mellitus in two patients, colon cancer in one pa¬ tient, and congenital hypogammaglobulinemia in one patient.

Congo Red Staining In Ibm

The Table shows the results of Congo red staining. All IBM cases demonstrat¬ ed intracellular Congo red, green-birefringent deposits that were heteroge¬ neous in appearance (Fig 1) . The green-biréfringent amyloid deposits varied in size, measuring 1 2 to 8 µ and rarely up to 20 µ in length. These deposits assumed different shapes; some were delicate and wispy appearing and others were more plaquelike. Occasional de¬ posits were intranuclear. In bright-field microscopy, the Congo red color ap¬ peared as a muted red compared with brighter red extracellular deposits, usually associated with the systemic amyloidoses.

Congo Red Staining Of Control Specimens

Control specimens included cases of dermatomyositis and polymyositis and a variety of vacuolar myopathies (Ta¬ ble). Chloroquine and acid maltase defi¬ ciencies are particularly notable for autophagic features that include whorls of membranous material, myelin figures, and secondary lysosomes.2' None of these cases showed amyloid deposits as seen in IBM. However, in rare autophagic vacuoles (irrespective ofthe disease) very small (250to 400-nm) green-birefringent granules were infrequently seen within the vacuoles. These differed from IBM deposits because of their smaller punctate appearance.

Five cases of hereditary vacuolar myopathies were evaluated. Four of these cases were similar to those in the family described by Kalimo et al,21' while the other was a case of vacuolar myopa¬ thy of skeletal and cardiac muscle. In a single muscle fiber, of more than 100 vacuolated fibers examined in these five cases, a linear, green-birefringent de¬ posit similar to that observed in IBM was seen. The nature of this deposit remains unknown and electron micros¬ copy of the specimens in these cases showed only autophagic vacuoles, but no filaments typical of IBM.

In addition, in a case of distal myopa¬ thy (showing rimmed vacuoles but no inflammation), congophilic, green-bire¬ fringent deposits were seen in the cyto¬ plasm and the muscle also showed 15to 18-nm filaments adjacent to vacuoles, identical to those seen in IBM. Similar filaments, appearing in distal myopa¬ thy, have previously been described.27

Quantitation Of Amyloid-Positive Fibers

In IBM, the amyloid deposits were restricted to vacuolated fibers and showed no relation to the inflammatory infiltrate typical of IBM. The deposits were usually seen adjacent to vacuoles, and less often perinuclear. Intranuclear amyloid deposits were seen rarely. The number of amyloid-positive fibers var¬ ied greatly between cases, but quantita¬ tive morphologic studies demonstrated a linear correlation with the number of Fig 1. -Vacuolated fibers in inclusion body myositis show Congo red (arrows in A and B) and corresponding green-birefringent intracellular amyloid deposits (C and D), respectively. Delicate wispy deposits (E) contrast to the plaquelike deposits (F) (bar In A = 20 µ ).

vacuolated fibers per biopsy (ri value for linear regression line is .892; five of 15 data points lie within 90% confidence level) (Fig 2) .

Immune And Histochemical Staining

Amyloid deposits in IBM retained their affinity for Congo red dye after incubation with potassium permanga-nate. This finding distinguishes the IBM amyloid fibril proteins from those observed in reactive (AA type) amyloidosis. showed no immune staining with antisera to human component, transthyretin, or immunoglobulin light chains.

Electron Microscopy The IBM filaments observed in all of our cases ranged in size from 15 to 20 nm with the majority measuring approximately 16 nm (16.6 ± 2.3 [mean ± SD], based on 25 random measurements). The maximum length of individual IBM filaments observed was 0.8 µ . To compare IBM filaments with those of systemic amyloidosis, we studied cardiac muscle laden with transthyretin amyloid deposits. The fil¬ aments appeared similar, but in cardiac amyloid the diameter was 8 to 13 nm (mean ± SD, 10.6 ± 1.77 nm) com¬ pared with IBM filaments measuring 15 to 18 nm (mean ± SD, 16.6 ± 2.3 nm) (Fig 3) .

Comment

In this study, we found Congo red, green-birefringent deposits especially in vacuolated muscle fibers in IBM.

These deposits are amyloid by defini¬ tion because of their affinity for Congo red dye and the characteristic green bi¬ refringence demonstrated on polariza¬ tion microscopy."12 The amyloid depos¬ its in IBM were heterogeneous, some delicate and wispy, and others more plaquelike in appearance. These intra¬ cellular collections of amyloid occurred adjacent to vacuoles, less often perinuclear, and rarely intranuclear. The dis¬ tribution corresponds to the typical lo¬ cation of the characteristic filaments, which are the hallmark and distinguish¬ ing feature of IBM.1"4 These findings strongly indicate that the amyloid de¬ posits seen by histochemical staining and IBM filaments are one and the same. This is further substantiated by the striking ultrastructural resem¬ blance of IBM to amyloid filaments. 11, 12 In fact, it was the morphologic similar¬ ity to amyloid filaments that served as a Fig 3. -Electron micrographs comparing amyloid filaments in inclusion body myositis (left) and cardiac amyloid (right). Bar = 0.5 µ . stimulus for us to examine IBM muscle biopsy specimens using Congo red dye.

Control specimens included other types of inflammatory myopathies and other vacuolar myopathies. No intracel¬ lular amyloid deposits were seen in cases of polymyositis or dermatomyosi¬ tis. This finding has practical implica¬ tions, indicating that alkaline Congo red staining can facilitate the diagnosis and differentiation of IBM from other in¬ flammatory myopathies. The advan¬ tages of Congo red staining are obvious when compared with the effort and ex¬ pense involved in searching for IBM fil¬ aments by electron microscopy. A note of caution, however, is that the number of positive fibers varies greatly be¬ tween cases (Fig 2) and the x 40 objec¬ tive is usually required to see these small intracellular deposits. A wide ar¬ ray of vacuolar myopathies were used as control samples. In one previously diag¬ nosed case of distal myopathy, showing no inflammation, typical green-birefringent deposits were found. This case also exhibited 15to 18-nm filaments by elec¬ tron microscopy. These filaments have been found in other cases of distal myop¬ athy,2' serving to blur the distinction between some types of distal myopathy and IBM. There was one additional case, however, of a hereditary vacuolar myopathy similar to the X-linked autophagic myopathy described by Kalimo et al26 that showed one positive muscle fiber. Of interest is that muscle biopsy specimens were available from two brothers and two additional cases of he¬ reditary vacuolar myopathies and none showed amyloid deposits. Further¬ more, electron microscopy failed to re¬ veal IBM filaments in this case. We do not have an explanation for this single positive fiber of more than 100 vacuolat¬ ed fibers examined; it most likely repre¬ sented an artifact. One additional cave¬ at for interpretation was the occasional appearance of green-birefringent punc¬ tate granules found within autophagic vacuoles irrespective of diagnoses (eg, acid maltase deficiency, IBM). The small size and punctate appearance al¬ lows them to be differentiated from IBM filaments.

Apart from the practical application of these findings, the most important question relates to the significance of amyloid filaments in IBM. Amyloid pro¬ teins are heterogeneous, and the specif¬ ic protein of origin varies with the dis¬ ease process. The intracellular location of amyloid deposits in IBM suggests a distinction from the proteins associated with systemic amyloidosis. Further im¬ mune staining failed to reveal any cross reactivity with human component (a nonspecific finding in many types of sys¬ temic amyloidosis), transthyretin, or immunoglobulin light chains. The loca¬ tion of IBM-associated amyloid favors a protein synthesized within the cell.

However, whether it represents an al¬ tered protein derived from a normal gene product or an isoform of a normal protein resulting from a gene mutation cannot be distinguished. For example, recent studies have shown that the 4.2-kd amyloid protein29"31 of Alz¬ heimer's disease is derived from a much larger amyloid precursor protein en¬ coded by a gene on chromosome 21.32'85 Antibody studies suggest that accumu¬ lations of amyloid precursor in second¬ ary lysosomes lead to proteolytic events that form the 4.2-kd amyloid peptide.36

The close association of IBM-associated amyloid with autophagic vacuoles also raises the possibility of a posttranslational modification of a normal muscle protein within an acidic degradative vacuolar compartment. Through such posttranslational modification, it is pos¬ sible that the end product might acquire resistance to proteases and become amyloidogenic.

Another consideration raised by the amyloidogenic properties of IBM fila¬ ments relates to the earlier proposal of Chou8 of an infectious cause for IBM.

Although no evidence supports that IBM is related to mumps virus,9,10 the filaments do share properties with prions, another class of infectious agent.18"16 Prions have been held ac¬ countable for the transmissible central nervous system neurodegenerative dis¬ orders, including scrapie of sheep and goats, and the human diseases, ie, kuru, Creutzfeldt-Jakob disease, and Gerstmann-Sträussler syndrome.13"16,37"39 Prusiner and colleagues13 demonstrated that purified scrapie prions, isolated from hamster brains, aggregated into rod-shaped particles measuring 10 to 20 nm in diameter, resembled amyloid by electron microscopy, and showed green birefringence using Congo red dye. Subsequent studies40"41 showed that infectivity was inseparable from a sialoglycoprotein of molecular weight 27 000 to 30 000, designated bodies raised to PrP 27-30 neutralized scrapie infectivity42 Furthermore, PrP 27-30 antisera not only localized within plaques of scrapie-infected hamster brain but also revealed filaments mea¬ suring 16 nm in diameter within the plaques.14 The diameter ofthe prion and IBM filaments are virtually identical, and both are slightly larger than sys¬ temic amyloid filaments.43 Additional similarities between IBM and human prion diseases are worthy of note. Approximately 5% to 15% of cases of Creutzfeldt-Jakob disease are famil¬ ial, and most cases of Gerstmann-Sträussler syndrome are inherited. 38, 39 Of particular interest is that both spo¬ radic and familial forms ofIBM occur.44,45

In fact, of the 24 subjects forming the basis for this report, two were brothers.

In addition, the recently reported famil¬ ial form of IBM affecting five of six male siblings was associated with periventri¬ cular leukoencephalopathy.45 In Gerstmann-Sträussler syndrome and familial Creutzfeldt-Jakob disease, accumulat¬ ing evidence supports both an infectious and a genetic basis related to PrP en¬ coded by a cellular gene.46"'" PrP genes have been sequenced in humans,50 vari¬ ous species of hamsters,01 mice,52,53 rats,54 and sheep.50 In scrapie disease, the infection appears to be linked to a PrP isoform that differs from control PrP because of a posttranslational mod¬ ification.56 In the human prion disease, Gerstmann-Sträussler syndrome, a point mutation of the PrP gene on chro¬ mosome 20 has been demonstrated at codon 102.47,48 The proline to leucine sub¬ stitution codes for an isoform of PrP that may be responsible for the disease.

Further studies will be required to establish the nature of IBM filaments.

The amyloidogenic properties provide new insight and suggest approaches not previously considered for this disease. The relation to human prion diseases is provocative but not by any means estab¬ lished. Identification of a transmissible agent will require approaches parallel¬ ing those employed for the studies of transmissible neurodegenerative dis¬ eases.10 Perhaps even more important¬ ly, identifying intracellular amyloid also raises possibilities of altered muscle proteins that accumulate within the cy¬ toplasm because of resistance to the usual intracellular digestive process. Such changes could be acquired or ge¬ netic. Of interest is the growing list of neurologic diseases arising from muta-tions in amyloidogenic proteins.57 No matter what final explanation accounts for the appearance of amyloid fibrils in IBM, the observations presented here provide clues to the pathogenesis of this poorly understood disorder, which have not previously been brought into focus.

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